Genetic inactivation of D-amino acid oxidase enhances extinction and reversal learning in mice

Abstract

Activation of the N-methyl-D-aspartate receptor (NMDAR) glycine site has been shown to accelerate adaptive forms of learning that may benefit psychopathologies involving cognitive and perseverative disturbances. In this study, the effects of increasing the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO), were examined in behavioral tests of learning and memory. In the Morris water maze task (MWM), mice carrying the hypofunctional Dao1(G181R) mutation demonstrated normal acquisition of a single platform location but had substantially improved memory for a new target location in the subsequent reversal phase. Furthermore, Dao1(G181R) mutant animals exhibited an increased rate of extinction in the MWM that was similarly observed following pharmacological administration of D-serine (600 mg/kg) in wild-type C57BL/6J mice. In contextual and cued fear conditioning, no alterations were found in initial associative memory recall; however, extinction of the contextual fear memory was facilitated in mutant animals. Thus, an augmented level of D-serine resulting from reduced DAO activity promotes adaptive learning in response to changing conditions. The NMDAR glycine site and DAO may be promising therapeutic targets to improve cognitive flexibility and inhibitory learning in psychiatric disorders such as schizophrenia and anxiety syndromes.

Figure 1.

D-serine concentrations in the brains of wild-type (+/+) and Dao1^G181R mutant (G181R/G181R) mice. HPLC was used to measure D-serine levels in whole brain and isolated brain regions including whole cortex, prefrontal cortex, hippocampus, amygdala, and cerebellum. n = 7–9 per group; *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-type mice, within the same brain structure.

Figure 2.

Dao1^G181R mutant mice have improved reversal memory in the MWM task. The path length (A, left panel) and latency (A, right panel) to reach a target platform were examined in wild-type (+/+) and mutant (G181R/G181R) mice during a visible platform session (day 1), an acquisition training phase (days 2–8), and a reversal training phase (days 10–12). In the acquisition (B) and reversal (C) probe trials, the percentage of time spent (left panels) and the number of crosses (right panels) over the target area were measured in wild-type and mutant animals. The dashed line represents chance level, corresponding to the ratio of the target area to the total pool area (2.6%). Platform locations include the reversal target (RT) in the NW quadrant, the acquisition target (AT) in the SE quadrant, and the nontarget areas in the NE and SW quadrants. n = 11 wild types and 15 mutants; *P < 0.05, **P < 0.01 compared with wild-type mice within the same platform location; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the reversal target within the same genotype.

Figure 3.

The Dao1^G181R mutation augments extinction responses in the MWM. The path length (A, left panel) and latency (A, right panel) to attain a target platform was evaluated in wild-type (+/+) and mutant (G181R/G181R) mice during the visible platform session (day 1) and acquisition training phase (days 2–8). Multiple probe trials were performed on alternate days (1–15), allowing extinction to be examined in the Dao1^G181R wild-type and mutant mice. In a control group of mutant mice, a probe trial was given only on day 9. During the probe trials, the time spent (B) and frequency of crosses (C) over the target area were determined. Chance levels are depicted as a dashed line. n = 8 wild types, 8 mutants, and 6 mutant controls; *P < 0.05, **P < 0.01 compared with wild-type mice within the same day; #P < 0.05, ##P < 0.01 compared with mutant mice that received several extinction trials.

Figure 4.

D-serine treatment enhances extinction rates in the MWM. Male C57BL/6J mice were treated with saline or D-serine (600 mg/kg) before each probe trial (alternate days 1–15), and extinction was evaluated in these mice. Also, a control group of mice was administered D-serine injections in the home cage (alternate days 1–7) and was given a probe trial only on day 9. The amount of time spent (A) and number of crosses (B) over the target area were determined for each treatment group. The dashed line indicates chance level. n = 9 saline, 8 D-serine, and 7 D-serine controls; *P < 0.05, **P < 0.01 compared with saline-treated mice within the same day; ##P < 0.01, ###P < 0.001 compared with D-serine-treated mice that received several extinction trials.

Figure 5.

Loss of DAO activity facilitates the extinction of contextual fear memory. Freezing responses in wild-type (+/+) and Dao1^G181R mutant (G181R/G181R) animals were measured during a training session (A) that contained a baseline activity period, followed by a 30-sec exposure to an auditory tone that coterminated with a foot shock (US). The percentage time spent freezing to the context (B) and to the tone in an altered context (C) was evaluated in wild-type and mutant mice during the extinction trials (days 1, 3, 5, 7, and 13). Freezing responses were also measured in a control group of mutants exposed to the context and tone on day 7 only. n = 14 wild types, 17 mutants, and 17 mutant controls; *P < 0.05 compared with wild-type mice within the same day; #P < 0.05 compared with mutant mice that received multiple extinction trials.